Regulated exocytosis by platelets, known as the release reaction, is critical to platelet function since platelets serve to respond to tissue damage by secreting components needed for hemostasis and tissue repair. While the signals eliciting the release reaction in platelets, and the materials released, are fairly well characterized, the actual mechanism by which dense core and alpha granules fuse with the surface-connected canalicular system is still not understood. The goal of this research proposal is to identify and characterize the proteins involved in mediating granule-target membrane fusion in the platelet. Recent advances in the understanding of synaptic vesicle fusion have suggested a paradigm for regulated exocytosis that will serve as the basis for this research proposal. The data suggest that the docking and fusion of vesicles to their target membranes is mediated by a neuron-specific set of integral membrane proteins (termed SNAP receptors or SNAREs) which specifically interact with a more generally expressed set of cytosolic proteins (SNAPs and NSF). It has been hypothesized that the matching of a SNARE from the vesicle (v-SNARE) with its cognate SNARE in the target membrane (t-SNARE) governs vesicular targeting. This docking complex serves as a binding site for the SNAPs which in turn mediate NSF binding to the membranes, completing the formation of the fusion complex. Although this mechanism is consistent with events leading to synaptic vesicle fusion, the generality of this hypothesis has yet to be fully tested in other exocytic systems. The experiments discussed in this proposal are directed toward the isolation, cloning, expression and characterization of potential v-SNARE and t-SNARE proteins from platelets. Anti-platelet SNARE antibodies will be generated in order to determine the subcellular localization of the SNARE proteins, and, utilizing an in vitro assay (also a goal of this proposal), these reagents will be used to study the SNAREs' role in the platelet release reaction. Using the SNAREs as ligands it may be possible to identify other proteins which, through their interaction with the SNAREs, may play a role in regulating exocytosis in the platelet. Through identification and characterization of the SNAREs of platelets, it should be possible to understand more about granule-plasma membrane fusion in the release reaction. This understanding will not only shed light on the mechanisms of initiating hemostasis but may lead to alternative methods for limiting thrombosis and for correcting the platelet-related defects in bleeding times seen in several different genetic diseases. To summarize, the proposal has 3 specific aims: i) Identify and/or clone SNARE-like proteins from platelets, and evaluate their tissue distributions, abilities to participate in fusion complex formation, phosphorylation and palmitoylation, and subcellular localizations, ii) Isolate and/or clone regulatory proteins that might control SNARE function and determine their specificities for specific SNARES, subcellular localizations, metabolic labeling and impact on fusion complex formation, and iii) Reconstitute platelet exocytosis in a permeabilized platelet system and use this system to evaluate specific protein requirements (identified in aims 1 and 2) for a-granule, dense granule, and lysosome secretion during platelet activation.